biologia plantarum

Biologia plantarum 45:533-538, 2002 | DOI: 10.1023/A:1022368924239

Ornithine Carbamoyltransferase from Spinacea oleracea: Purification and Characterization

E. Bellocco¹, C. Di Salvo¹, G. Laganà¹, A. Galtieri¹, S. Ficarra¹, A. Kotyk², U. Leuzzi¹

¹ Department of Organic and Biological Chemistry, University of Messina, Messina, Italy

² Institute of Physiology, Academy of Sciences of the Czech Republic, Prague 4, Czech Republic

Ornithine carbamoyltransferase (OCT) from spinach (Spinacea oleracea L.) was purified to homogeneity and studied for some kinetic and structural properties. The enzyme showed a specific activity of 436 U mg^-1, its molecular mass was approximately 118 kDa as estimated by Sephacryl S-200 gel filtration chromatography, the purified protein ran as a single band of 38 kDa in sodium dodecyl sulfate-polyacryamide gel electrophoresis. The enzyme catalyses an ordered bi-bi-sequential reaction in which carbamoyl phosphate binds first, followed by L-ornithine; L-citrulline leaves first, followed by phosphate. The Michaelis constant was 0.19 mM for L-ornithine and 13.1 µM for carbamoyl phosphate; the dissociation constant for the enzyme and carbamoyl phosphate complex was of 19 µM. The K_m of the reaction decreases from pH 6.0 to pH 10.4. The enzyme is heat-labile, but it was protected from thermal inactivation by substrates; more by ornithine alone than by two substrates acting together.

Keywords: enzyme kinetics; enzyme purification; δ-N-(phosphonoacetyl)-L-ornithine; spinach

Subjects: ornithine carbamoyltransferase, purification, characterization, spinach; δ-N-(phosphonoacetyl)-L-ornithine; Spinacea oleracea

Published: December 1, 2002  Show citation